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Developing a robust ultrafiltration-LC-MS/MS method for quantitative analysis of unbound ASA404 in human plasma

Li, Wenkui and Lin, Hui and Smith, Harold and Tse, Francis (2011) Developing a robust ultrafiltration-LC-MS/MS method for quantitative analysis of unbound ASA404 in human plasma. Journal of Chromatography B, 879 (1). pp. 1927-1933. ISSN 1570-0232

Abstract

Ultrafiltration of human plasma in combination with LC-MS/MS has been increasingly used in the quantitative analysis of the free fraction of drug candidates for PK/efficacy assessment. In addition to controlling the pre-incubation and centrifugation temperatures, several important factors that must be investigated and addressed include: (1) ultrafiltration device selection, (2) possible nonspecific binding, (3) possible impact of freeze/thaw cycles of plasma samples and extended storage of plasma samples at room temperature on the analyte recovery prior to ultrafiltration, and (4) identification of the appropriate assay dynamic range to avoid unnecessary dilutions. These issues were explored in the development and validation of a robust LC-MS/MS assay for the quantitative analysis of unbound ASA404 in human plasma. First, to mimic human physiological conditions, all plasma samples were incubated at ~37C for a minimum of 30 minutes after thawing and prior to centrifugation to obtain the ultrafiltrate. Second, by passing the calibration standards and QC samples in plasma ultrafiltrate through the ultrafiltration membrane, the observed non-specific binding of the analyte due to the membrane was corrected. Third, the effects of multiple freeze/thaw cycles and/or storage at room temperature for various periods (4, 8, 16 and 24 h) were evaluated to determine the impact on analyte concentrations in the ultrafiltrate from the plasma QC samples. Fourth, the appropriate dynamic range was established to accommodate the expected incurred sample free analyte concentrations. The validated assay has a dynamic range of 30.0 to 30000 ng/ml for ASA404 in human plasma ultrafiltrate using a sample volume of 30 µl. Quality control pools containing the analyte were prepared at concentrations of 30.0 to 22500 ng/ml to cover the assay calibration range. The intra-assay and inter-assay precision and accuracy were  15% (CV) and within ±15% (bias) of the nominal values, respectively, for all QC concentrations, including the LLOQ. Freeze/thaw for up to three cycles of the plasma samples and/or the extended human plasma sample exposure to room temperature for up to 24 hours were confirmed to have no impact on the assay results for the free analyte. The validated method was successfully implemented to support clinical studies for the compound.

Item Type: Article
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Additional Information: author can archive post-print (ie final draft post-refereeing); Publisher's version/PDF cannot be used
Keywords: ASA404, ultrafiltration, plasma, unbound analyte, free analyte, LC-MS/MS, nonspecific binding, stability, incurred sample stability, incurred sample reanalysis.
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Date Deposited: 13 Oct 2015 13:15
Last Modified: 13 Oct 2015 13:15
URI: https://oak.novartis.com/id/eprint/4365

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